The erythropoietin (Epo) receptor is a model for understanding signal transduction mechanisms of the cytokine receptor superfamily. Calcium signaling plays an important role in cell proliferation and apoptosis, yet little is known about regulation of ion channels by hematopoietic growth factors. Using quantitative fluorescence microscopy and electrophysiology, our laboratory was the first to demonstrate that Epo modulates the intracellular calcium concentration ([Ca2+]i) through activation of members of the TRPC ion channel family. The transient receptor potential (TRP) superfamily is a diverse group of voltage-independent calcium permeable channels which are involved in sustained calcium entry in nonexcitable cells. TRPC3 and TRPC6 are members of the TRPC subfamily which are expressed in human erythroid cells. TRPC3 is activated by Epo, while TRPC6 inhibits TRPC3 activation. TRPC2 is also activated by Epo in murine erythroid cells but is a pseudogene in humans. In this grant, Specific Aim 1 will study the mechanisms through which Epo regulates [Ca2+]i by activation of TRPC3. We will examine (1) the physiological importance and mechanisms regulating TRPC3 tyrosine phosphorylation after Epo stimulation by identifying the involved kinase and key tyrosine sites on TRPC3 which are phosphorylated and required for channel activation;(2) the role of phospholipase C and IP3R in TRPC3 activation by Epo;and (3) the mechanisms through which Epo regulates TRPC3 cell surface localization. We will utilize a two phase liquid culture system of primary human erythroid progenitors/precursors to study mechanisms of regulation of TRPC3 and their role in human erythropoiesis. Specific Aim 2 will examine the mechanisms through which TRPC6 inhibits TRPC3 activation by Epo and the physiological importance. TRPC3 and TRPC6 expression are regulated during normal erythroid differentiation. We will determine whether TRPC6 inhibits TRPC3 membrane localization or tyrosine phosphorylation, and identify TRPC6 domains which are involved in inhibition of TRPC3. Specific Aim 3 will examine the functional importance of TRPC3 in erythropoiesis using TRPC3 whole animal and tissue specific knockout mice, and TRPC2-/-TRPC3-/- double knockouts. Preliminary results show that TRPC3 knockout animals have a defect in red cell production under stress. Erythropoiesis will be characterized in TRPC3-/- and TRPC2-/-TRPC3-/- mice, which replicate TRPC3 deficiency in the human environment. Understanding the role of TRP channels in erythroid proliferation and differentiation is of fundamental biological importance. In addition, modulation of TRPC3 may have important applications in future therapy of diseases of red cell production including polycythemia and certain anemias, such as that of chronic renal failure. PUBLIC HEALTH RELEVANCE: This research is among the first to study regulation of an ion channel, TRPC3, by a hematopoietic growth factor, erythropoietin, and our studies with knockout mice suggest that TRPC3 is important in the production of red blood cells. The work that we propose has broad relevance in understanding and treating disease of decreased red cell production secondary to many causes including anemia of chronic renal failure, as well as diseases of overproduction such as polycythemia or erythroleukemia. Therapeutic manipulation of TRPC3 activity may have wide applicability in regulating blood cell production, and hence have direct relevance in improving public health.